Long Interspersed Nuclear Elements (LINEs) are a class of retrotransposable elements that continually mutate genomes, including mammalian genomes. The currently active family of LINEs in humans is called LINE-1 (L1). To replicate, L1 mRNA and two L1-encoded proteins (called ORF1p and ORF2p) form a cytoplasmic ribonucleoprotein particle (RNP) intermediate. The L1 RNP enters the nucleus and reverse transcribes L1 mRNA at new chromosomal sites. This process, called retrotransposition, has been spectacularly successful in humans and has generated over one-third of the human genome. In addition, expression of L1 RNPs in humans and/or model organisms is associated with mutations, DNA damage, aging, and various disorders, such as cancer, infertility, and neurologic disease. In most of these cases we do not know whether L1 is simply correlated with these disorders, or if L1 plays a causative role in these disorders. Our inability to convincingly address this question is due to a gap in our rudimentary understanding of LINE biology, which limits our ability to manipulate endogenous L1 activity in a specific manner. The simplicity of a typical LINE element belies the complexity of LINE retrotransposition, suggesting that LINE RNPs utilize host proteins (henceforth referred to as ?host factors?) to assist in their replication. Although some host factors that interact with L1 RNPs have been identified, almost all of these factors are RNA-binding inhibitors that block retrotransposition, or are artifacts of non-specific RNA binding. We still have little knowledge of the core host factors that are crucial for a successful round of LINE retrotransposition. This bias towards retrieving L1 inhibitors is likely due to methodology, and alternative strategies are needed for identifying core LINE host factors. A long term goal in our lab is to address this gap ? we want to understand how LINEs replicate, specifically by identifying and characterizing LINE RNPs/host proteins interactions important for active retrotransposition. To this end, we have developed a yeast model of LINE retrotransposition, based on the Candida albicans L1-like element called Zorro3. We have already used this yeast model to discover that the endosomal sorting complex required for transport (ESCRT) physically interacts with LINE RNPs and is critical for retrotransposition in both yeast and humans. In this proposal we will further characterize this interaction by determining where, how, and why the L1/ESCRT interaction takes place (Aim 1). We have also discovered that LINE retrotransposition occurs in a phenotypically distinct state of Candida albicans called the opaque state. This finding is the basis for a novel proteomics strategy (Aim 2) to identify biologically relevant host factors that interact with LINE RNPs. The strategy in Aim 2 addresses a major shortcoming of previous studies ? identifying the biologically significant host factors from the sea of host proteins that bind to L1 RNPs in biochemical pulldowns.